Recent advances in nucleic acid testing (NAT) technologies have been aimed at isothermal methods to enable point-of-care testing (POCT) of infectious and genetic diseases. These NATs have led to improvements in enzyme-based nucleic acid amplification strategies, which have been used to detect DNAs and RNAs in prokaryotic and mammalian cells without thermocycling. Although these strategies offer the potential to develop new bioanalytical assays for SNP (single nucleotide polymorphism) detection without thermocycling, these methods typically require enzymes, multiple primers, nucleotides, and extension reactions, which limit the utility for POCT where minimal sample preparation, low cost, and rare mutation detection are key considerations. Specifically, enzyme inhibitors are found in many laboratory samples and clinical specimens, such as whole blood, urine, saliva, and CSF, which significantly increases the risk of false negatives, resulting in ambiguities around a diagnosis. Moreover, extension reactions are error-prone and could lead to inaccurate results. Additionally, co-amplification of non-target sequences without enriching the target sequence in heterogeneous samples can affect the limit of detection (LOD) for rare mutations in clinical specimens. From a cost perspective, enzymes and nucleotides significantly increase the cost of a reaction, and multiple primers add complexity and a need for repeated redesign-evaluation cycles, and hence cost to each reaction. In addition to these limitations, NATs are too slow to enable rapid POC stratification. PCR and next-generation sequencing (NGS) technologies require enzymes, nucleotides, thermocycling, extension reactions and have a LOD of only 0.1% for rare mutations, and therefore face similar challenges to those discussed above for NATs for POCT.
Accordingly, there is a need for a new technology with a low LOD and/or high sensitivity in genetically heterogeneous samples to minimize false-negative test results, enable early detection of infectious and genetic disease when therapeutic interventions are much more effective, and enable routine genetic screening in the general population, both in clinical and POC settings. Furthermore, a new technology that enables faster time-to-results would provide added benefits to treatment management, such as 1) post-operative routine monitoring of residual disease after surgical debulking for disseminated cells; 2) routine screening in doctors' offices with greatly reduced testing cycles; and 3) self-administered tests without waiting for doctor's visits.